Figure 6.

Tyrosine 719 associated p85α, SHP2, and Gab2 complex is sufficient for KITD814V-induced ligand-independent growth and survival in vitro and MPD in vivo. (A) Primary HSC/Ps derived from W^sh/sh mice, which lack endogenous expression of KIT receptor, were transduced with the indicated chimeric KIT receptors and sorted to homogeneity based on EGFP expression. Cells were starved in serum- and growth factor-free media for 6 hours and subjected to proliferation assay in the absence of growth factors by thymidine incorporation. Bars represent the mean (CPM ± SD) thymidine incorporation from 1 of the 3 independent experiments performed in quadruplicate. *P < .05, WT KIT vs KITD814V. **P < .05, KITD814V vs KITD814V-F7. ***P < .05, KITD814V-F7 vs KITD814V-Y719. (B) The 32D cells bearing the indicated chimeric receptors were starved of serum and growth factors and cultured for 48 hours in the absence of growth factors. Cells were harvested and stained with anti–annexin V antibody and 7-amino-actinomycin D followed by flow cytometric analysis. Double-negative cells in the lower left quadrant are indicated as surviving cells. Representative dot blots are shown. (C) Kaplan-Meier survival analysis of mice transplanted with the primary HSC/Ps bearing indicated KIT receptors (n = 8-18 per group). Results show that restoration of Y719 alone is sufficient to induce transformation in vivo (median survival, 55 days; n = 15). (D) The 32D cells bearing the indicated chimeric KIT receptors were starved in serum- and growth factor-free medium for 8 hours. Starved cells were lysed, and equal amounts of protein lysates were subjected to Western blot analysis using an anti–phospho-KIT (Y719), anti–phospho-AKT, total AKT, Bcl-x_L, and β-actin antibodies as indicated. Similar results were observed in 3 independent experiments.